Various publications, including patents, published applications and scholarly articles, are cited throughout the specification. Each of these publications is incorporated by reference herein, in its entirety. Citations not fully set forth within the specification may be found at the end of the specification.
Coffee aroma and flavor are key components in consumer preference for coffee varieties and brands. Coffee's characteristic aroma and flavor stems from a complex series of chemical reactions involving flavor precursors (Maillard reactions) that occur during the roasting of the bean. Flavor precursors include chemical compounds and biomolecules present in the green coffee bean. To date, over 800 chemicals and biomolecules have been identified as contributing to coffee flavor and aroma (Montavon et al., 2003, J. Agric. Food Chem., 51:2328-34; Clarke & Vitzthum, 2001, Coffee: Recent Developments. Blackwell Science).
Because coffee consumers are becoming increasingly sophisticated, it is desirable to produce coffee with improved aroma and flavor in order to meet consumer preferences. Both aroma and flavor may be artificially imparted into coffee products through chemical means. See, for example, U.S. Pat. No. 4,072,761 (aroma) and U.S. Pat. No. 3,962,321 (flavor). An alternative approach would be to use techniques of molecular biology to either add aroma and flavor-enhancing elements that do not naturally occur in coffee beans, or to enhance those elements responsible for the flavor and aroma that are naturally found in the coffee bean. Genetic engineering is particularly suited to achieve these ends. For example, coffee proteins from different coffee species may be swapped. In the alternative, the expression of genes encoding naturally occurring coffee proteins that positively contribute to coffee flavor may be enhanced. Conversely, the expression of genes encoding naturally occurring coffee proteins that negatively contribute to coffee flavor may be suppressed.
The endogenous coffee proteins whose expression could be the target of genetic manipulation, and whether and to what extent production of such coffee proteins should be enhanced or suppressed has been empirically determined. The 11S storage protein has been identified as one such candidate coffee protein. (Montavon et al., 2003, J. Agric. Food Chem. 51:2335-43). Coffee oleosin, because of its role in oil storage, is another candidate coffee protein. Coffee oils are known constituents of coffee aroma and flavor. For example, (E)-2-nonenal, and trans-trans-2-4-decadienal are lipid derived volatiles important to coffee aroma (Akiyama et al., 2003; Variyar et al., 2003). Therefore, increasing or decreasing the stores of these oils in the coffee bean should have a measurable effect on the aroma and flavor of the coffee. Oleosins also form lipid bilayers and may contribute to lipid content as well.
Oleosins have been detected in a variety of plant species including oilseed rape, (Keddie et al., 1992), african oil palm (NCBI), cotton (Hughes et al, 1993), sunflower (Thorts et al., 1995), barely (Aalen et al, 1994; 1995), rice (Wu et al., 1998), almond (Garcia-Mas et al., 1995), cacao (Guilloteau et al., 2003) and maize (Qu and Huang, 1990; Lee and Huang, 1994). In plant seeds, oil bodies, also called oleosomes, are maintained by oleosins. These oil bodies are thought to serve as a reservoir of triacylglycerols (TAG) (Tzen et al., 1993). One function of oleosins is to organize the lipid reserves of seeds in small, easily accessed structures (Huang et al., 1996). Seed oil bodies range in diameter from 0.5 to 2 μM (Tzen et al., 1993), providing a high surface to volume ratio, which is believed to facilitate the rapid conversion of TAGs into free fatty acids via lipase mediated hydrolysis at the oil body surface (Huang et al., 1996). In seeds containing large amounts of oils, such as oilseed rape, oleosins represent 8%-20% of the total protein (Li et al., 2002) and oleosins represent 79% of the proteins associated with arabidopsis oil bodies (Jolivet et al., 2004). Oleosins cover the surface of these oil bodies (Huang, 1996), where they are thought to help stabilize the lipid body during desiccation of the seed by preventing coalescence of the oils. Related lipid containing particles are also found in certain specialized cells. For example, the tapetum, a structure involved in the development of pollen; also has specific oil body-like lipid particles called tapetosomes. These oil body-like particles are involved in providing functional components required for microspore and pollen development (Murphy et al., 1998; Hernandez-Pinzon et al., 1999).
Oleosin proteins are composed of three distinctive domains: a central conserved hydrophobic fragment of approximately 72 amino acids flanked by a highly variable N-terminal carboxylic motif and a C-terminal amphipathic α-helix (Huang, 1996; Li et al, 2002). The lengths of the amino and carboxy portions are highly variable, and as a consequence, oleosins can range in size from 14 to 45 kDa (Tai et al., 2002; Kim et al., 2002). The amphipathic amino and carboxylic portions allow the protein to reside stably on the surface of the oil bodies (Huang, 1996). The amino acids at the center of the hydrophobic region contain three conserved prolines and one conserved serine, which form the proline KNOT Motif. This motif is believed to allow the central fragment to fold into a hydrophobic hairpin, which anchors the oleosin in the oily central matrix (Huang, 1996). The role of the proline KNOT motif on protein function was further investigated by Abell et al. (1997) who showed that, if the three proline residues were substituted by leucine residues, an oleosin-beta-glucuronidase fusion protein failed to target to oil bodies in both transient embryo expression and in stably transformed seeds.
Oleosins have been classified as high or low-Mr isoforms (H- and L-oleosin) depending on the relative molecular masses (Tzen et al, 1990). Sequence analysis showed that the main difference between the H- and L-oleosins was the insertion of 18 residues in the C-terminal domain of H-oleosins (Tai et al., 2002) and Tzen et al. (1998) have shown that both forms coexist in oil bodies. In Zea mays, Lee and Huang (1994) identified three genes, OLE16, OLE17 and OLE18 with molecular weights of 16, 17 and 18 kDa, respectively, that are expressed during seed maturation. The corresponding protein ratios are 2:1:1 respectively in isolated oil bodies (Lee and Huang, 1994; Ting et al., 1996). Lee et al. (1995) classed OLE16 as an L-oleosin and OLE17 and OLE18 as H-oleosins, indicating that oil bodies of Z. mays contain equal amounts of H- and L-oleosins in oil bodies. Furthermore, the oil bodies of rice embryos were found to contain a similar amount of two distinct oleosins of molecular masses 18 and 16 kDa corresponding to the H form and L-form respectively (Tzen et al., 1998; Wu et al., 1998). Two oleosins were also identified in the seeds of Theobroma cacao (Guilloteau et al., 2003). At 15 and 16.1 kDa these proteins represent one L-form and one H-form respectively.
Kim et al. (2002) have characterized the oleosin genes in Arabidopsis into three groups. The first group consists of oleosins expressed specifically in the seeds (S), the second expressed in the seeds and the floral microspores (SM) and the final group expressed in the floret tapetum (T). Of the sixteen oleosin genes identified in the Arabidopsis genome, five genes were shown to be specifically expressed in maturing seeds, three genes expressed in maturing seeds and floral microspores and eight in the floral tapetum (Kim et al., 2002). The five seed specific oleosins of Arabidopsis have been previously classed as 3 H-form oleosins and 2 L-form oleosins by Wu et al. (1999). Sesame, maize and rice have all been shown to encode three seed-specific oleosins (Tai et al., 2002; Ting et al., 1996; Chuang et al., 1996; Wu et al., 1998; Tzen et al., 1998).
Oleosin expression is believed to be developmentally and spatially regulated, primarily at the level of transcription (Keddie et al., 1994). Wu et al. (1998) showed that transcripts of two rice oleosins appeared seven days after pollination and vanished in mature seeds. A similar result was obtained by Guilloteau et al. (2003) who showed that the level of the two cacao oleosin transcripts decreased in mature seeds. While oleosin gene transcription has been studied in a semi-quantitative manner in a number of seed types, there are no reports in which the transcript levels of most, or all, of the oleosins in one seed type have been quantitatively determined during seed development.
Despite the fact that coffee grains have an oil content of between 10 and 16%, little is known about oleosin proteins in coffee. There is a dearth of scientific data regarding the number of coffee oleosins, their protein structure, their expression levels and distribution throughout the coffee plant and among coffee species, their oil storage capabilities, and the regulation of their expression on the molecular level. Thus, there is a need to identify and characterize coffee oleosin proteins, genes, and genetic regulatory elements. Such information will enable coffee oleosin proteins to be genetically manipulated, with the goal of improving one or more features of the coffee, including oil content and stability, which in turn can affect roasting parameters, ultimately impacting the aroma and flavor of the coffee.
For purposes of enhancing or suppressing the production of coffee proteins such as oleosins, it is desirable to have available a set of promoters compatible with the coffee plant. In addition, any genetic manipulation should ideally be localized primarily or solely to the coffee grain, and should not adversely affect reproduction or propagation of the coffee plant.
Seed-specific promoters have been described. Examples of such promoters include the 5′ regulatory regions from such genes as crucipheran (U.S. Pat. No. 6,501,004), napin (Kridl et al., Seed Sci. Res. 1:209:219, 1991), phaseolin (Bustos et al, Plant Cell, 1(9):839-853, 1989), soybean trypsin inhibitor (Riggs et al., Plant Cell 1(6):609-621, 1989), ACP (Baerson et al., Plant Mol. Biol., 22(2):255-267, 1993), stearoyl-ACP desaturase (Slocombe et al., Plant Physiol. 104(4):167-176, 1994), soybean a′ subunit of beta-conglycinin (P-Gm7S, Chen et al., Proc. Natl. Acad. Sci. 83:8560-8564, 1986), Vicia faba USP (P-Vf.Usp, U.S. patent application Ser. No. 10/429,516). In addition, a Zea mays L3 oleosin promoter has been described. (P-Zm.L3, Hong et al., Plant Mol. Biol., 34(3):549-555, 1997).
Seed-specific promoters have found application in plant transformation. For example, groups have used genetic manipulation to modify the level of constituents of seeds. See, Selvaraj et al., U.S. Pat. No. 6,501,004, Peoples et al. U.S. Pat. No. 6,586,658, Shen et al., U.S. patent application Ser. No. 10/223,646, Shewmaker et al., U.S. patent application Ser. No. 10/604,708, and Wahlroos et al., U.S. patent application Ser. No. 10/787,393. Of note is that oleosin promoters have been used successfully in these systems.
However, seed-specific promoters, and more specifically, coffee oleosin promoters heretofore have not been used in the transformation of coffee plants. Thus, there exists a need to have available additional gene regulatory sequences to control the expression of coffee proteins. In the same vein, there exists a need to have available gene regulatory sequences to control the expression of oleosins in coffee plants. Furthermore, there exists a need to have available gene regulatory sequences to control the expression of coffee proteins in the coffee grain. In this regard, promoters specific to gene expression in the coffee grain are highly attractive candidates, among these promoters are coffee oleosin promoters.